Sp1 transcription factor Abstract In the ayurvedic system of medicine, the medicinal plant, Withania somnifera Dunal (Solanaceae)

finds application for numerous ailments including cancer. This herbal plant yields a host of steroidal lactones called withanolides, some of which have shown growth inhibition of human tumor cell lines. Withaferin A amongst these withanolides reportedly is very active in impairing antitumor activity. However; the underlying molecular mechanisms of this activity remains still unclear. In the present study, we have shown that withaferin A inhibited vascular endothelial cell growth factor (VEGF) -induced tube formation by human umbilical vein endothelial cells (HUVECs) and angiogenesis in chick chorioallantoic membrane (CAM) assay. In Ehrlich ascites tumor (EAT) model, the animals treated with withaferin A suppressed in vivo, the peritoneal angiogenesis and microvessel density. When compared to the untreated animals, the withaferin A treated tumor bearing mice showed a decrease in the volume of ascites and tumor cell number. Quantitation of VEGF levels in ascites from withaferin A untreated or treated tumor bearing mice indicated decreased secretion of VEGF in ascites from treated mice, as measured by ELISA. Studies at molecular level revealed that withaferin A inhibits binding of Sp1 transcription factor to VEGF-gene promoter, in Withaferin A suppresses the expression of vascular endothelial growth factor in Ehrlich ascites tumor cells via Sp1 transcription factor Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath* Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore-570006, India. For Correspondent : Salimathuom@rediffmail.com order to exert its antiangiogenic activity. These results clearly indicate the antiangiogenic potential of withaferin A in modulating antitumor activity. Keywords: Ehrlich ascites tumor; Withaferin A; Angiogenesis; Sp1, VEGF. Introduction Several natural compounds are recognized as cancer chemo preventive agents. Withanalides are especially well known to suppress tumor cell growth via cell-cycle arrest and by the induction of apoptosis in several tumor cell lines (1-3). Moreover, withaferin A inhibits endothelial cell proliferation and angiogenesis in vitro (4). Angiogenesis is essential for the growth, progression and metastasis of solid tumors (5). Withaferin A, a member of the withanalides family that is present at high levels in roots and leaves of Withania somnifera plant has been found to possess antioxidant and antitumor activity (6-9). However, the mechanism by which withaferin A suppresses angiogenesis has not been fully elucidated.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that facilitates tumor growth and metastasis. Hypoxia is known to induce the expression of VEGF gene (10, 11). VEGF promoter analysis has revealed several potential transcription factor-binding sites, such as hypoxia-inducible factor-1(HIF-1), activator Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 139 Prasanna et al protein (AP)-1, AP-2, early growth response1(Egr-1) and Sp1 (12). The GC box-binding protein, Sp1 is a ubiquitous transcription factor that belongs to the Sp family of transcription factors, consisting of Sp1, Sp2, Sp3, and Sp4 (13). Sp1 has been implicated in the transcription of large number of genes, including housekeeping genes, tissuespecific genes and genes involved in growth regulation (13-15). Sp1 activities are regulated by a variety of stimuli. Most of these regulations occur through either post-translational modification or alteration of Sp1 protein abundance. The principal known post-translational modifications are phosphorylation and glycosylation through the O-linkage of the monosaccharide, N-acetylglucosamine (OGlcNAc) (16). Expression level of the VEGF mRNA is tightly regulated by both transcriptional and posttranscriptional mechanisms. Recent studies have demonstrated that intracellular signaling pathways and genetic elements are involved in controlling its expression. VEGF promoter activity is preceded by the activation of transcription factor Sp1 (17). Therefore it is clear that a constitutive Sp1 activation is essential for the differential over expression of VEGF, which in turn plays an important role in angiogenesis and the progression of cancer. It has also been shown that Sp1 in particular, plays an important role in tumor angiogenesis and contributes to the aggressive nature of human pancreatic adenocarcinoma (18). In this study, we tested the hypothesis that the antiangiogenic effect of withaferin A on EAT cells involves a reduction in secretion of ascites fluid and expression of VEGF, which is regulated by Sp1 transcription factor. Moreover, we investigated the molecular mechanism by which withaferin A inhibits angiogenesis in vivo. Materials and methods Materials Ehrlich ascites tumor (EAT) cells were routinely maintained in Swiss albino mice in the animal house, University of Mysore, Mysore, India. Endothelial growth medium (EGM-2) was procured from Cambrex Biosciences, Walkersville, USA. Dulbeccos modified Eagles

medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were purchased from Invitrogen, USA.T4 polynucleotide kinase kit was obtained from Amersham biosciences. The Sp1 oligonucleotides (5-d (ATTCGA TCG GGG CGG GGCGAG C)3) for gel shift assays were obtained from Promega. Radioacive -[32P] ATP was obtained from Bhabha Atomic Research Centre (BARC), Mumbai, India. RNA isolation kit was procured from Qiagen, USA. Secondary antibodies conjugated to alkaline phosphatase and proteinase inhibitors were obtained from Bangalore Genei, Bangalore, India. The rest of the chemicals were of analytical grade of purity and were procured locally. Methods Isolation of withaferin A from Withania somnifera roots Withania somnifera roots were collected locally from Mysore, India. The plant specimens were identified and authenticated at the herbarium of the Department of Botany, University of Mysore, Mysore, India. The roots were washed, shade dried and powdered. One hundred grams of the root powder was extracted in 500ml of methanol overnight. Withaferin A was isolated from the methanol extract of Withania somnifera roots as previously described (4). The compound Withaferin A (10mg) was dissolved in 100l of DMSO and diluted 100 times with sterile distilled water to make final concentration 1g/l and used for subsequent experiments. Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 140 Sp1 transcription factor Human Umbilical Vein Endothelial Cells (HUVECs) culture HUVECs were purchased from Cambrex Biosciences, Walkersville, USA. The cells were cultured in 25 cm3 tissue culture flask (NUNC, Genetix Biotech Asia, Bangalore, India) and grown using EGM-2 medium and endothelial cell basal medium according to the manufacturers protocol. Incubation was carried out in a humidified atmosphere of 5% CO2 in air at 370C. When cells reached confluency, they were passaged after trypsinization. HUVECs of passages 2-5 were used for the experiments. Animals and in vivo tumor generation Six to eight weeks old mice were acclimated for one week while caged in groups of five. Mice were housed and fed a diet of animal chow and water ad libitum throughout the experiment. All experiments were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. EAT cells (5106cells/

mouse) injected intraperitoneally grow in mice peritoneum forming an ascites tumor with massive abdominal swelling. The animals show a dramatic increase in body weight over the growth period and the animals succumb to the tumor burden 15-16 days after implantation. The number of cells increased over the 14 days of growth with formation of 7-8 ml of ascites fluid with extensive neovascularization in the inner lining of peritoneal wall. EAT cells from fully grown tumor bearing mice were harvested from the peritoneal cavity of mice (19). The ascites fluid was collected in isotonic saline solution containing 3.8% sodium citrate. The cells were pelleted by centrifugation (3000 rpm for 10 min at 40C). Contaminating red blood corpuscles if any were lysed with 0.8% ammonium chloride. Cells were resuspended in 0.9% saline. These cells or their aliquots were used either for transplantation or for further experiments. Tube formation assay Tube formation of HUVECs was conducted for the assay of in vitro angiogenesis. The assay was performed as described in earlier report (20). Briefly, a 96-well plate was coated with 50l of Matrigel (Becton Dickinson Labware, Bedford, MA), which was allowed to solidify at 370C for 1 hour. HUVECs (5x 103 cells per well) were seeded on the Matrigel and cultured in EGM medium containing withaferin A (3.5-14g) for 8 hours. After incubation at 370C and 5% CO2, the enclosed networks of complete tubes from five randomly chosen fields were counted and photographed under an Olympus inverted microscope (CKX40; Olympus, New York, NY) connected to a digital camera at 40X magnification. Chick chorioallantoic membrane (CAM) assay CAM assay was carried out according to the detailed procedure as described by Gururaj, A.E. et al. (21, 22). In brief, fertilized chicken eggs were incubated at 370C in a humidified incubator. On the 11th day of development, a rectangular window was made in the egg shell and glass cover slips (6-mm diameter) saturated with 25ng/ml vascular endothelial growth factor (VEGF) and VEGF + withaferin A (7g) was placed on the CAM and the window was closed using sterile wrap. The windows were opened after 48h of incubation and were inspected for changes in the microvessel density in the area below the cover slip and photographed using a Nikon digital camera. In vivo withaferin A treatment inhibits EAT growth To determine whether withaferin A inhibits tumor growth and angiogenesis in EAT cells in vivo, withaferin A (7mg/kg/day/mouse) and vehicle control (0.1% of DMSO) was injected

into the EAT bearing mice every alternate day Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 141 Prasanna et al after 6th day of tumor transplantation and growth of the tumor was monitored by taking the body weight of the animals. Animals were sacrificed on the 14th day and the EAT cells along with ascites fluid were harvested into the beaker and centrifuged at 3000 rpm for 10 min at 40C. The pelleted cells were counted by Trypan blue dye exclusion method using a haemocytometer. A measure of the supernatant gave the volume of ascites fluid. Peritoneal angiogenesis and micro vessel density After harvesting the EAT cells from control and withaferin A-treated animals, the peritoneum was cut open and the inner lining of the peritoneal cavity was examined for extent of neovasculature and photographed. Formaldehyde fixed and paraffin embedded tissues of peritoneum from EAT bearing mice either treated or untreated with withaferin A were taken and 5m sections were prepared using automatic microtome (SLEE Cryostat) and stained with hematoxylin and eosin. The images were photographed using LeitzDIAPLAN microscope with CCD camera and the blood vessels were counted. Quantitation of VEGF EAT bearing mice were treated with or without withaferin A (7mg/kg/day) for 5 doses on 6th, 8th, 10th and 12th day of tumor transplantation. The animals were sacrificed and ascites fluid was collected after 24h of each dose. VEGF-ELISA was carried out using the ascites fluid (21, 23, 24). In brief, 100l of ascites from tumor bearing mice either with or without withaferin A treatment, was coated using coating buffer (50 mM carbonate buffer pH 9.6) at 40C overnight. Subsequently, wells were incubated with anti-VEGF165 antibodies, followed by incubation with secondary antibodies tagged to alkaline phosphatase and detection using p-nitrophenyl phosphate (pNPP) as a substrate. Preparation of nuclear extracts Nuclear extracts were prepared according to the method previously described (25). Briefly, cells (5X106) treated either with or without withaferin A in complete HBSS for different time intervals were washed with cold phosphate buffered saline and suspended in 0.5 ml of lysis buffer (20mM HEPES, pH 7.9, 350 mM NaCl, 20% Glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM EGTA, 0.5 mM DTT, 1 mM Pefablock, 1g/ml Aprotinin, 1g/ ml Leupeptin). The cells were allowed to swell on ice for 10 min; the tubes were then vigorously mixed on a vortex mixer for 1 min and centrifuged at 10,000 rpm for 10 min at

40C. The supernatant was immediately stored at -200C. Electrophoretic Mobility Shift Assay (EMSA) Nuclear proteins were extracted from EAT cells treated either with or without withaferin A for 60,120 and 180 min respectively. The EMSA was performed as described in earlier report (26, 27). The double stranded Sp1 consensus oligonucleotide probes [5-d (ATT CGA TCG GGG CGG GGC GAG C)-3] were end-labeled with -[32P] ATP. Nuclear proteins (40g) were incubated with 40fmoles of -[32P]-labeled Sp1 consensus oligonucleotides for 30min in binding buffer containing 100mM HEPES (pH 7.9),10mM MgCl2, 125 mM KCl, 0.5mM EDTA, 4% glycerol,0.5% NP-40,1g of poly [dI-dC] and 1mg/ml BSA. The samples were electrophoresed in 4% non denaturing polyacrylamide gel in 0.5% TBE at room temperature for 2 hr at 200V. The gel was dried, transferred to imaging plate (IP) and the image was scanned by image analyzer Fujifilm (FLA-5000). Results Withaferin A inhibits tube formation of HUVECs induced by VEGF In order to verify if withaferin A interferes directly with the formation of blood vessels by HUVECs, we performed tube formation assay Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 142 Sp1 transcription factor in vitro. The HUVECs were plated on the Matrigel. The HUVECs in the basal medium could not form tubes and VEGF was used to induce the tube formation. In the positive control group stimulated with VEGF (10ng), HUVECs rapidly aligned with one another and formed tube-like structures resembling a capillary plexus within 8 hours, after VEGF treatment. However, treatment with withaferin A prevented VEGF stimulated tube formation of HUVECs in a concentration (3.5-14g) - dependent manner (Fig. 1). Meanwhile, no cytotoxicity was observed under this concentration range of withaferin A used in the assay. Withaferin A was shown to interfere with the ability of HUVECs to form the in vitro vessel-like tubes, one of the important traits of the endothelial cells. Withaferin A inhibits VEGF induced neovascularization on chick chorioallantoic membrane CAM assay is one of the widely used validation assays for formation of new blood vessels. In order to further verify if withaferin A is an inhibitor of new blood vessel formation, withaferin A was applied on chorioallantoic membrane of chick embryo to test its in vivo antiangiogenic activity. In the CAM assay model withaferin A induced avasculature zone formation in the developing embryos. Notably newly formed

microvessels were regressed around the area of withaferin A treated CAM (Fig. 2). Fig. 1: Effect of withaferin A on VEGF induced HUVECs tube formation HUVECs (5X103 cells) cultured in EGM medium with 3.5g, 7g and 14 g withaferin A was added to the Matrigel coated 96 wells plate. After incubation for 8 hours at 370C, capillary networks were photographed and quantified (Magnification: X40). Concentration dependent inhibition of tube formation by withaferin A was recorded. All datas are presented as mean from three different experiments with triplicates and means of S.E.M. P<0.05 vs control. Fig. 2: Effect of withaferin A on neovascularization in the chick CAM Withaferin A was applied on CAM of 11-dayold chicken embryo. After 48h of incubation, the applied area was inspected for changes in neovascularization. The arrows indicate the applied area. The data shown represents the result of an experiment, which was done using maximum six eggs in each group. All photographs were taken at same magnification. Withaferin A inhibits growth of EAT cells and secretion of ascites in vivo Initially, proliferation of tumor cells in mice was monitored by measuring the weight of the animals every day. A decrease in body weight in withaferin A treated animals was observed when compared to the increased body weight of the untreated tumor bearing mice. It was also observed that withaferin A lessened the tumor burden considerably in a dose dependent manner showing the optimum activity at 7mg/kg/dose. Cell number was counted after each dose of withaferin A treatment. In control group, which is tumor bearing mice not treated with withaferin A, the number of EAT cells increased exponentially. In Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 143 Prasanna et al contrast, in the withaferin A treated group, the number of cells were drastically decreased (Fig. 3A). This implied that withaferin A inhibit tumor cell growth in vivo. The volume of ascites was also measured using ascites from EAT bearing as well as withaferin A treated EAT bearing animals. The results indicated that withaferin A treatment reduces the secretion of ascites fluid (Fig. 3B). It is indicative from this data that withaferin A is probably capable of inhibiting the proliferation of tumor cell growth in vivo. Fig. 3: Effect of withaferin A on EAT cell number and ascites volume in vivo EAT cells (5X106 cells/animal, i.p.) were injected into mice. After 6 days of tumor transplantation, withaferin A (7mg/kg/animal) was injected on days 7th, 9th, 11th and 13th. The animals were sacrificed on days 8th, 10th, 12th and 14th. EAT cells were collected along with ascites fluid.

Cells were counted in haemocytometer (A) and ascites volume was measured (B). At least five mice were used in each group and the results obtained are an average of three individual experiments and means of S.E.M. Fig. 4: Withaferin A inhibits angiogenesis and microvessel density A) Inhibition of peritoneal angiogenesis. EAT bearing mice were treated with and without withaferin A for four doses (7mg/kg/animal). The mice were sacrificed and the peritoneal lining was observed for extent of neovascularization. We presented representative photograph of peritoneum. B) Reduction in microvessel density (MVD) The peritoneum of control as well as withaferin A treated EAT bearing mice was embedded in paraffin and 5m sections were taken using microtome. The sections were stained with hematoxyline and eosine and observed for microvessel density. Arrows indicate the microvessels. Withaferin A inhibits angiogenesis in vivo Sprouting of new blood vessels is evident in the inner peritoneal lining of EAT bearing mice. The peritoneal lining of EAT bearing animals and withaferin A treated mice was inspected for the effect on tumor-induced peritoneal neovascularization. EAT bearing mice treated with withaferin A showed decreased peritoneal angiogenesis when compared to the extent of peritoneal angiogenesis in untreated EAT bearing mice (Fig. 4A). Histopathological staining of peritoneum sections from the EAT bearing group appeared well vascularized. In contrast withaferin A treated peritoneum sections were characterized by a pronounced decrease in micro vessel density and the caliber of detectable vascular channels. While tumor bearing peritoneum sections showed 17 1.2 blood vessels, withaferin A treated peritoneum showed 6.8 1.3 blood vessels (Fig. 4B). Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 144 Sp1 transcription factor Withaferin A inhibits VEGF secretion in ascites fluid of EAT bearing mice In order to verify whether withaferin A induced inhibition of neovascularization and microvessel density is due to decreased secretion of VEGF by EAT cells, we have quantified the secreted VEGF in ascites fluid of control and different doses of withaferin A treated animals by ELISA. It is evident in Fig. 5 that withaferin A inhibits production of VEGF in EAT cells. In EAT bearing mice, 1280ng of VEGF was detected in ascites. However in case of withaferin A treated animal ascites 220ng of VEGF was detected per mouse. performed. The results indicated that withaferin A inhibits binding of Sp1 transcription factor to the proximal promoter region of the VEGF gene.

In contrast, there was prominent band showing the binding of Sp1 to the proximal promoter region (Fig. 6) when nuclear extract from tumor bearing mice was used. Fig. 5: Effect of withaferin A on in vivo production and expression of VEGF EAT bearing mice were injected with withaferin A (7mg/ kg/animal) for four doses and ascites fluid was collected after sacrificing the animal every alternate day after each dose of treatment. ELISA was carried out to quantitate VEGF in ascites fluid using antiVEGF165 antibodies. Animals bearing EAT cells not treated with withaferin A was used as control. Withaferin A inhibits Sp1 DNA binding activity in EAT cells To verify for the involvement of transcription factor Sp1 in withaferin A induced inhibition of VEGF gene expression, an electrophoretic mobility shift assay was Fig. 6: Effect of withaferin A on Sp1-DNA binding activity Nuclear extracts were prepared from EAT cells untreated and treated with withaferin A. Sp1-DNA binding activity was assayed by EMSA using Sp1 oligonucleotides. Discussion Plant and dietary products contribute to about one-third of potential anticancer drugs and the preventive effects of plant-based diets on tumorigenesis and other chronic diseases have been well documented (28). Withania somnifera (L.) Duna1 commonly known as Ashwagandha (family Solanaceae) is extensively used in many indigenous preparations. W. somnifera is reported to have anti-inflammatory (29), antiarthritic (30) and anti-tumor (31) activities. Withaferin A, a withanolide was isolated and reported to be antiangiogenic and anti-tumor active Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 145 Prasanna et al principle from Withania somnifera. A recent study demonstrated that the anti-angiogenic effect of withaferin A was due to the inhibition of endothelial cell proliferation (4). However, the detailed molecular mechanisms involved in the anti-angiogenic effect of withaferin A were not clearly understood. In this study we investigated the anti-angiogenic effects of withaferin A both in vitro and in vivo model. Withaferin A suppressed human endothelial cell- tube formation which is one of the hallmarks of angiogenesis indicating that withaferin A inhibits endothelial cell proliferation. This may be due to the induction of HUVECs apoptosis by withaferin A (4). Further, in Ehrlich ascites tumor bearing mice and also by using several ex-vivo and cell based validation assays, we observed that withaferin A besides inhibiting the growth of the tumor suppressed

peritoneal angiogenesis and microvessel density by down regulating VEGF gene expression and VEGF secretion into the ascites of tumor bearing mice. It also inhibited neoangiogenesis induced by VEGF in CAM assay indicating that withaferin A targets both tumor and endothelial cell to exert its anti proliferative and antiangiogenic activities. Increased VEGF expression is closely associated with an increase in microvessel density (32). VEGF being a permeability factor plays fundamental role in the fluid accumulation and tumor growth in ascites tumor. By secreting VEGF, ascites tumor enhances the permeability of preexisting microvessel lining of peritoneal cavity to stimulate ascites formation thereby tumor progression. Inhibition of fluid accumulation, tumor growth and microvessel density by neutralization of VEGF has been demonstrated underlying the importance of VEGF in malignant ascites formation (33-35). Our results indicated that there was decrease in the VEGF secretion in withaferin A treated animals. Inhibition of VEGF secretion could be due to inhibition of activity of transcription factors NF-B, AP-1 or Sp1 which are involved in the regulation of VEGF gene expression. Withaferin A is already reported as a potent inhibitor of NF-B and AP-1 DNA binding activity (4, 36, 37). Recent studies indicated that Sp1 transcription factor plays an important role in VEGF expression and tumor angiogenesis. A region between nucleotide-109 and -61 of the VEGF promoter and its intact Sp1-binding sites were required for the inhibition of VEGF promoter activity. In this study, we found that withaferin A treatment reduced Sp1 DNA binding activity to the proximal promoter region of VEGF gene in a time dependent manner. It was shown recently that celecoxib inhibits VEGF expression and reduces angiogenesis and metastasis of human pancreatic cancer via suppression of Sp1 (38). Sp1 suppression was closely correlated with reduced VEGF level. Withaferin A has been reported as potent inhibitor of PKC and TNF dependent IB kinase , which subsequently blocks NF-B nuclear translocation (3, 37). PKC isoforms are also involved in the activation of Sp1, NF-B and AP-1 in B16F1 murine melanoma cells (39, 40). In summary, our experiments have shown that inhibition of VEGF secretion and tumor microvessel formation is one of the potential mechanisms by which withaferin A suppresses the growth of Ehrlich ascites tumor. Additionally, the suppression of VEGF secretion appears to be as a consequence of altered Sp1 transactivation and inhibition of VEGF gene expression. The data clearly indicates a novel mechanism for the antiangiogenic activity of withaferin A and also substantiate the important

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